This study uses the well-established 4T1.2 and 67NR mammary cancer cell lines as syngeneic immunocompetent implantable murine models of triple negative breast cancer.

Using pair-wise analysis, we compare the implantation route of cancer cells by conventional orthotopic injections in the mammary fat pad vs intraductal injection in the mammary ductal tree. We reveal that implantation methodology influences the inherent metastatic potential, tumour progression and the co-evolution of cancer cells and the tumour microenvironment.

Intraductal injection accelerates tumour growth, especially in the non-metastatic 67NR model, and better mimics early tumourigenic events that preserve the tissue microenvironment crucial for tumour-stromal interactions. Immune cell composition differences are driven by the tumour model but independent of the implantation method, however, intraductal implantation carries higher stromal proportions at early stages of tumour development. In the 4T1.2 model, intraductal injection increases metastatic burden. We found markedly different systemic inflammation traits unique to the metastatic 4T1.2 scenario consistent with the acquisition of a cancer immunotolerant phenotype during progression to metastatic disease. Metastatic dissemination induced a myeloid-dominated inflammatory milieu, particularly driven by granulocytic myeloid-derived suppressor cells, contrasting with the lymphocyte-rich profile in the non-metastatic 67NR tumours. Immune responses of the metastatic 4T1.2 scenario were further analysed by targeted single-cell RNA sequencing revealing distinct immune activation in primary tumours, emphasising the differential role of immune cells between primary and metastatic lesions.

Our findings stress the importance of carefully selecting experimental models to faithfully recapitulate breast cancer progression and metastasis, providing insights for future therapeutic interventions focused on immune modulation.